LOCUS       sequence-196808-        2962 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Backbone for cloning guides that are compatible with LwaCas13a. 
            Contains a 5' direct repeat. Clone using BbsI (BpiI). F overhang 
            AAAC. R overhang AAAA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2962)
  AUTHORS   Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung 
            J, Zhang F
  TITLE     RNA editing with CRISPR-Cas13.
  JOURNAL   Science. 2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180.
  PUBMED    29070703
REFERENCE   2  (bases 1 to 2962)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science. 
            2017 Oct 25. pii: eaaq0180. doi: 10.1126/science.aaq0180."
COMMENT     Sequence Label: pC0040-LwaCas13a crRNA backbone
FEATURES             Location/Qualifiers
     source          1..2962
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     259..278
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     512..529
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    646..667
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(682..687,688..705,706..712)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    720..736
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     725..747
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     744..760
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     744..760
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        774..1022
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     774..794
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     945..964
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1106..1123)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1106..1122)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1115..1137)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1331..1350)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1450..1472
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1510..1528)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1596..1700
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1701..1769,1770..2561)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1919..1938)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(2732..2962,1..358)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt
       61 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga
      121 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct
      181 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg
      241 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca
      301 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa
      361 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt
      421 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
      481 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga
      541 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      601 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct
      661 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat
      721 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccagagggcc
      781 tatttcccat gattccttca tatttgcata tacgatacaa ggctgttaga gagataattg
      841 gaattaattt gactgtaaac acaaagatat tagtacaaaa tacgtgacgt agaaagtaat
      901 aatttcttgg gtagtttgca gttttaaaat tatgttttaa aatggactat catatgctta
      961 ccgtaacttg aaagtatttc gatttcttgg ctttatatat cttgtggaaa ggacgaaaca
     1021 ccgatttaga ctaccccaaa aacgaagggg actaaaacat gtcttcctgg gacgaagaca
     1081 attttttggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa
     1141 accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta
     1201 atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat
     1261 ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt
     1321 gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa
     1381 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     1441 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     1501 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
     1561 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt
     1621 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat
     1681 aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt
     1741 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg
     1801 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga
     1861 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc
     1921 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac
     1981 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg
     2041 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca
     2101 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg
     2161 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg
     2221 acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg
     2281 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag
     2341 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg
     2401 gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct
     2461 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac
     2521 agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact
     2581 catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga
     2641 tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt
     2701 cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
     2761 gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc
     2821 taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc
     2881 ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc
     2941 tcgctctgct aatcctgtta cc
//
